Announcements Columbus Day 10/8 No class, 10/9 BU Monday!

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1 Announcements Columbus Day 10/8 No class, 10/9 BU Monday! Make up section for Tuesday discussion: Wednesday 10/10, 5-6 pm, SAR 102 Monday Section: Tuesday 10/9, am KCB 106 Wednesday Section: Wed. 10/10, am SAR 300 No quiz in any section next week

2 Chapter 3 Week 2 Parts D,E Purification of Lactate Dehydrogenase (LDH) Purpose: Understand the effects of ionic strength on proteins and precipitate proteins via ammonium sulfate Use dialysis to de-salt a protein sample

3 Protein Solubility Depends on number of hydrophilic and hydrophobic residues on protein surface Majority of hydrophobic residues on inner globular core of protein Hydrophilic residues tend to stay on outer surface to interact with aqueous solution Charged and polar groups more likely to form salt bridges and hydrogen bonds Also depends on properties of the solution in which the protein is dissolved Blue = hydrophillic White = hydrophobic

4 Major Factors Influencing Protein Ionic Strength Stability Many ions effect stability of other ions in solution ph Extremes tend to have poor protein stability Temperature Extremes tend to have poor protein stability Presence of Denaturants SDS, Urea, Guanidinium chloride, [SCN - ], etc. Necessary in some complex mixtures Dielectric Constant of the Solvent Relative polarity of solvent

5 Ionic Strength Effects Salts have different effects on proteins depending on ionic strength Protein solubility increases with neutral salts at low ionic strength Salting-in Protein solubility decreases with neutral salts at high ionic strength Salting-out Salting-in stabilizes charged groups of proteins Salting-out is competition between protein and salt for waters of hydration As salt concentration increases protein molecules aggregate and some fall out of solution

6 Ammonium Sulfate Precipitation Method that allows us to use the relative ionic strength of different proteins to purify individual proteins Different proteins precipitate at different levels of ionic strength due to different secondary and tertiary structure Ammonium sulfate used in protein purification and crystallography to help salt-out proteins Need to watch ph % Saturation is unit used to denote ionic strength Salted-out proteins are separated after salt addition by centrifugation

7 Method to separate solvents from the rest of the protein Dialysis Semi-permeable membrane allows salt and solvent molecules out Protein molecules remain inside membrane Water diffuses in as salt diffuses out Selectively permeable membrane Large Enzyme Molecules Small salt/solvent molecules [Salt] inside and outside diffuse to equilibrium Buffer switched 2-3 times [Salt] from ~4 M to 4 μm

8 Add (NH 4 ) 2 SO 4 (aq) to 40% Sat. Centrifuge 12krpm, 10 min 1S Supernatant CRUDE EXTRACT Flow Chart for LDH Purification 2P Pellet Extraneous proteins 2S Supernatant LDH + Other proteins Add (NH 4 ) 2 SO 4 (s) to 75% Sat. Centrifuge 12krpm, 10 min 3P Pellet LDH + Other proteins 3S Supernatant Extraneous proteins Dialysis 2 Buffer exchanges by TF s 3P Dialyzed Affinity Purification Ultrafiltration PURE LDH! See flow chart p. 70

9 Week 2: Procedure Ammonium Sulfate Precipitation Activity Assays Protein Concentration via Dye Binding Dialysis Keep everything on ice! Especially LDH extract!

10 Week 2: Procedure Ammonium Sulfate Precipitation Thaw protein from week 1, save 1 ml aliquot Set up buret with saturated (NH 4 ) 2 SO 4 solution Bring to 40% Saturation Add 33 ml (NH 4 ) 2 SO 4 (aq) for 67 ml of crude extract Use appropriate proportion Add dropwise in beaker, and let equilibrate 5 min after adding Centrifuge 10 min at 12,000 rpm After Spin: 2S and 2P Where is LDH? Resuspend 2P in 15 ml of buffer SAVE ALIQUOTS OF EVERYTHING!

11 Week 2: Procedure Ammonium Sulfate Precipitation Measure volume of 2S Add solid (NH 4 ) 2 SO 4 to bring final solution to 75% See table p. 65 (21.2 g/100 ml) Add while stirring over ~15 min Check ph midway through, ~7-8, adjust with NH 3 (aq) Equilibrate 5 min after addition Centrifuge 10 min at 12,000 rpm After Spin: 3S and 3P Where is LDH? Resuspend 3P in 15 ml of buffer SAVE ALIQUOTS OF EVERYTHING!

12 Activity Assays Week 2: Procedure Do LDH Activity Assays on: 1S Compare to week 1 activity 2S 2P No dilution 3S No dilution 3P Use 1 ml aliquots for assays All assays need to be in range of ΔA 340 /min of What do you use to blank your spectrophotometer?

13 Protein Concentration Dye Binding Assay Make new standard curve if necessary Find protein concentration for: 1S 2S 2P 3S 3P Week 2: Procedure Use 1 ml aliquots for protein concentration A 595 should be within linear region of your standard curve Dilute protein when necessary What do you use to blank your spectrophotometer?

14 Week 2: Procedure Dialysis TF s will show to how prepare bag Put all but 1 ml aliquot of 3P sample in dialysis bag Leave some head space at top of bag for expansion during dialysis Label tape in clamp with initials and leave same labeled tube with TF s TF s will switch dialysis buffer at least twice before your next lab and then will freeze your protein sample 3P Dialyzed Fraction

15 Activity Calculation [Activity] = Units/ml = μmol of Substrate Consumed or Product Formed min * ml [Activity] = ΔC = ΔA 340/min / ε app in mm -1 = (0.05/min)/(6.21 mm -1 ) = units/ml in the assay You must account for the dilutions of your protein! [Activity Undiluted ] = (ΔC)(Total Volume of Assay)(Dilution Factor) (Volume of enzyme used in assay) [Activity Undiluted ]= ( units/ml)(3.0 ml)(400) = 193 units/ml (0.05 ml)

16 More Enzyme Calculations Total Activity = (Activity)(Total Volume) = Units/ml* ml = Units Protein = Mass Protein/Volume Extract = mg/ml Total Protein = (Protein)(Total Volume) = mg/ml* ml = mg Specific Activity = Total Activity/Total Protein = Units/mg % Yield = Total Activity in Given Step Total Activity in Crude Extract x 100 Remember to account for the dilutions of your protein! For needed calculations, see purification table, p. 86

17 Purification Table Calculations Fraction Volume (ml) Corrected Volume (ml) Activity (units/ ml) Protein (mg/ml) Total Activity (units) Total Protein (mg) Specific Activity (units/ mg) Yield (%) ΔA340/ min Dilution Factor Homogenate S S P S P P-D Pooled Conc Only used 80 ml of original homogenate, therefore everything is multiplied by 2 Only loaded 9 ml for 5000 units for affinity column, therefore volume is multiplied by 4 (8 ml*4 = 32 ml)

18 Final Example Purification Table Fraction Corrected Volume (ml) Homogenate 160 Activity (units/ml) Protein (mg/ml) Total Activity (units) Total Protein (mg) Specific Activity (units/mg) Yield (%) 1S S P S P P-D Pooled Conc

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